Process for obtaining vitamink compounds



Patented Dec. 21, 1948 UNITED STATES PATENT OFFICE PROCESS FOR OBTAINING VITAMIN. K

COMPOUNDS Edward A. Doisy, Webst er Groves, and Sidney A.

Thayer, St. Louis, 'Mo., Donald W. MacCorquodale, Highland ,Park, 111.,

Ralph W. McKee,

Newton, Mass.,,and Stephen B. Binkley Detroit, Mi c h., assignors to President & Board of Trustees of St. Louis Un iversity,.S t. Louis, Mo.

-No .D rawing. Application May 11, 1944,

.Serial No. 535,204

1939,,now-Patent' No. [2,737,105, dated April .10,

1945,,and Serial No. 294,316, filed September 1, iesanonrat nt No. 2,3 48,037, dated Mas/.2, 1944.

It is knownv that vitamin K, pro ducts' and concentratesareobtainable. from various animal; and

vegetable materials, such as alfalfa; spinach, soy

been oil,ilivergputrifled, fishmeal, etc. n the othenhand, fit .is' also. known that th antihemorrhagic vitamins are labile substances which arespbieot. to inactivationin greater" pr-lsser amounts during the, usual 'processesof extraction,

adsorption, dlstillatio'n, eta, used. for isolating L hee,-

Ema-M 5 91; inre iqn ve s ines t0 "t, tent theobjections.-mentionedin the processes heretofore used, by reacting the vitaminK in its relativelycrude form with an' esterifyipg or etheriesi seetth u in 2 af e duct n o quinone groups to hydroquinone groups, to ob tion into the ester or ether, and use the. latter for tainan-ester oran etherof the reduced form. of

the; vitamin, which, because it is more, stable, than er ts-min. i t en u jec to t kn i m t O odsof concentration and. purification with practically no loss of activity. After the reduced and esterifled, .or. etherified, derivative has been, suflicientlyzconcentrated, itcan be converted back into -a purifledvitamin. product b'y'hydrolysis and oxidation.. Insteadof converting the derivative back .into Ithevitamin fromwhichl it was obtained, the

derivative can, in many instances, be directly used for. therapeutic purposes, .since it has. been:

found that the. derivatives, often have ,high antihemorrha ic potency, in some cases thaving ac,- tivityof the.. same.order,as,the original vitamin itself.

Theinvention,isnotlimited tothe use, of crude on impurervitamin .Kproducts as starting ma-- terials, .sinceitis possible tostart with .a pure 1rom. ,a.. natural,.-source, oriby synthesis, convert vi amin :K., as, obtained for. example by, isolation its vitamin K effect. An advantage in such cases isithat the ether or ester obtained has greater stability than the, natural vitamin from which it ,was derived.

Vitamin K activity is exhibited bya number of different substances. However, apparently all of them have a quinone-like chemical structure, and are capable of reduction to hydroquinonelike compounds. The present invention can therefore be more fully understood by considering the esterification or etherification step. of the present invention as a replacement of phenolic hydroXyl' groups in the reduced or hydroquinone form ofthe vitamin by groups capable of hydrolysis to give hydroxyl, such as -Oacy1,

The invention can be illustrated by the following examples:

Example 1 The starting material for this example is vitamin K1, a pure antihemorrhagic vitamin obtained by petroleum ether extraction of dried alfalfa meal and subsequent purification by adsorption on a zeolite, as described for example in J. A. C. S., 61, 1295. Such a product is hereinafter designated as .vitaminKi. It is. 2 methyl-3-phytyL-1, 4-naphthoquinone and has the following formula:

' on; CH3 on; CH3,

I I I CHCH=C GHzCHnGHgCHCHzCHiCHzCHC HzCHzCHzCHCHa Vitamin K1 is an oil at ordinary temperatures,

havinga polyene structure which absorbs 4 moles of hydrogen upon catalytic reduction, exhibits ultraviolet absorption spectrum maxima (hexane solution) at 243, 248, 261, 270, and 323m and has an antihemorrhagic potency of approximately 1 chick unit per 0.5 microgram.

milligrams of thisvitaminKi are dissolved along with 100 mgbof fused sodium acetate in 10 cc. of acetic anhydride. The solution is then refluxed with 1 gram of zinc dust for 30 minutes. An additional 1 g. of zinc dust is added, a few milligrams at a time, during the period of heating. ..:'I'he mixture is filtered while hot. The acetic anhydride filtrate is decomposed by the addition of. 20 to 30 cc. of water. After decompositionhas taken place,;the mixture is extracted with ether, the ether extract is washed .with water and evaporated to dryness. The residue is then taken of fine snow white needles from methyl alcohol or petroleum ether. It has a melting point of 59 C. and is diacetyl dihydro vitamin K1. Analysis of the compound gives the following figures:

Carbon, 78.21% and 78.01%. v Hydrogen, 10.07% and 10.03%.

Oxygen, 11.72% and 11.96% (by difference) Molecular weight, 531 (East) Microhydrogenation of a sample of the diacetyl dihydro vitamin K1 causes it to take up 3.04 moles of hydrogen (H2). The vitamin K1 itself took up 4.08 moles of hydrogen.

Bio assay by the chick method shows that the diacetyl dihydro vitamin K1 has an activity of about 500 units per mg.

Absorption spectrum examination of the diacetate of this example shows a general absorption in the region from 220 m to beyond 300 III/1. with intense absorption at 230 m where the extinction coefficient is OG O Ha -C Hr phytyl Diacetate of 2-methyl-3-phytyl-1, 4-naphthohydroquinone.

The diacetyl ple is converted to vitamin K1 by treating an anhydrous ether solution of the diacetyl dihydro compound with a 14 times excess of methyl magdihydro vitamin K1 of this exama melting 1 fractions and passing nesium iodide, adding water to hydrolyze to dihydro vitamin K1, and shaking an ether solution of the latter with air to oxidize it to the corresponding quinone-like "compound, vitamin K1.

After evaporating ofi the ether, the residue is fractionally distilled at 2 10- mm. pressure. About 90% of the vitamin activity is obtained in the fraction distilling at 115-145 C. This fraction is practically pure vitamin K1 and analyzesas follows:

Carbon, 82.34%. Hydrogen, 10.13%. Oxygen, 7 53% (by difierence) example is identical with the pure vitamin K1- started with is further proved by the fact that reductive acetylation can be repeated on it to give a diacetate of a dihydro vitamin having a by hydrolyzing' "200 mg. of fused sodium acetate in 2200. of acetic melting point and giving results in a bio-assay identical with the original diacetate of dihydro vitamin K1. The diacetate from-thesecond reductive acetylation also does not-give any JIOWGT- ing of the melting point in a mixed melting point test with the original diacetate of dihydro vitamin K1.

Example 2 The starting material for this example is a petroleum ether extract of dried alfalfa leaf containing about 100 chick units of vitamin K1 per milligram of solids. 250 mgs. of this concentrate are dissolved with 250 mgs. of fused sodium acetate in 25 cc. of acetic anhydride. The solution is then refluxed with 3 gms. of zinc dust for 30 minutes. An additional 2 gms. of zinc dust is added, a few milligrams at a time, during the period of heating. The mixture is filtered while hot, the acetic anhydride filtrate is decpmposed by "the addition of -90 cc. of water. j Afterthe anhydride has been decomposed, the mixture s extracted with ether, the ether extract washed and evaporated to dryness.

The residue is then t ,ken up in 100 .cc. ofper" troleum ether having a boiling point of 110 'C. and passed through a column .of adsorbent such. as Permutit or charcoal. One can use adsorbents. such as alumina, fullers earth, calcium barf-1;; bonate, etc., which are known tocause' extensive destruction of vitamin activity'when used with vitamin K1 itself. Using a columnof Permutit 35 mm. in diameter by 40 cm. long, the cc. of petroleum ether solution of thefcrude' diacetate of dihydro vitamin K1. is poured onto thetop of the column, followed successively by about 2 liters. of petroleum ether, 1%.; liters of petroleum ether containing 10% of benzene, l lite'r of pej-f' troleum ether containing 20% benzene, '1 liter' of petroleum ether containing 50% benzene, 2 liters" benzene and 1 liter alcohol, col1ecting'500 cc. fractions at the bottom of the column. The puri fled diacetate; of dihydrovitamin K1 is." fo'u'nd chiefly in the benzene fractions while most iof; the inactive material is obtained in earlier fra tions. The solvent is removed from the active fractions, and the residue is taken up'in and crystallized at low temperature 'from absolute' methyl alcohol. After recrystallizing a few -tiniesit melts at 59 C. and is identical with the diacetate of Example 1. By combining the least active them through the column again it is possible to obtain practically quantitativerecovery of all the vitamin activity in the form of substantially pure diacetate of dihydro vitamin K1.

Example 3 Material for this example: is designated-as" vitamin K2 and is a crystalline productmelting at 52 C. and obtained by extracting putrified fish meal with an organic solvent and treatingthe' extract with a zeolite adsorbent, as described-in A. C. S. 61, 1295. Vitamin K2 is a 2 methyl-3 alkyl-l, -naphthoquinone in whichsaid 3 alky group has 30 carbon atoms and 6 double bonds" 200 mg. of vitamin K2 is dissolved along-with anhydride. The solution is refluxed with 2 gm. of zinc dust for 3-0 minutes, adding an additional gram'of zinc dust, a fewmilligrams at a time, f

during the period of heating. The hot'mixture is filtered and the acetic anhydride' filtrate decom posed by adding 50 cc. of water. After the anhy dride has reacted with the water, the mixture is swam-:

extractedrwithsetherm the i etheraextractawashed with;:.:water andaevaporatedsto dryness; --';Thef resi:

due is taken up in:andvrecrys-tailized from cold absolute methanolwandthe diacetate'of dihydro vitamin 'Kzseparates asa whitecrystalline product melting at 57-53%0.

The diacetate"-giVes' the" following analysis I. Quinone formal-vitamin Esterification Mild oxidation innaccordance: withathez-present inventiombyrany; ofi'the-known'methods ofconcentration and p'uri-e' fication, such as adsorption, fractional distillation, fractional crystallization; etc., with-practically no loss-act vita-min activity.

Although the-examples showsimultaneous re-* duction andesterifi'c'ation of the vitamin; the l in vention also embodies the variation wherein the quinone-like v'itaminis first -reduced and thenester-ifiedor etherified. The two variationsofthe 1. process can be represented diagrammatically as follows 1 Diestenolthe hydroquinone form ot-vitamin K Concentrate lactivity- Hydrolyze Hydroquinone form of vitamin K Reduction II. Quinone form of vitamin K Hydroquinone form Mild 1 oxidation Hydroquinoue form of vitamin K Upon microhydrogenation, the diacetate takes up 7.99 mols. of hydrogen. Vitamin K2 itself takes up approximately 9 mols. of H2. The diacetate of dihydro vitamin K2 assays approximately 300 chick units per mg. Ultraviolet absorption is very similar to that of the diacetate of Example 1. It has an extinction coeflicient of Purified diester Esterii'y or Diester or diether of hydroquinone form Etherii'y Concentrate 1 activity CH; CH3 CH3 CH5 Example 4 This example is carried out as described under Example 2, but using as starting material a crude petroleum ether extract of putrified fish meal containing 120 chick units of vitamin K2 active material per milligram of solids. After the solvent is evaporated off of this extract, the crude vitamin K2 residue is converted to the crude dihydro diacetate by reductive acetylation and then purified by an adsorbent as described under Example 2.

The diacetate products of Example 2 and of Examples 3 and 4 can be hydrolized and oxidized to get back pure vitamin K1 and vitamin K2 respectively, in the same manner as described under Example 1. Instead of using sodium acetate and acetic anhydride, other alkali metal carboxylates and carboxylic acids can be used. For example, by using a propionic acid, benzonic acid, chloroacetic acid, or succinic acid anhydride, along with the corresponding alkali metal salt, one can obtain the dihydro vitamin propionates, chloroacetates, succinates, etc., such as 2-methyl-3- phytyl-l, 4-naphthohydroquinone dipropionate (dihydro vitamin K1 dipropionate), 2-methyl-3- phytyl-l, 4 naphthohydroquinone dibenzoate and succinic acid esters of 2-methyl-3-phytyl-l, 4-naphthohydroquinone and the alkali metal salts thereof;

The examples show that vitamin K which has been converted to its phenolic form and esterfied or etherified is more stable than its original quinone-like form. It can thereafter be treated,

What we claim as our invention is:

1. Process for obtaining an antihemorrhagic compound comprising treating an extract containing a crude form of a compound of the class consisting of vitamins K1 and K2 with a reducing agent and an agent of the class consisting of esterifying and etherifying agents thereby obtaining a crude form of dihydro diester of said vitamin, treating said crude diester product by known methods to obtain a substantially pure diester and converting the latter by hydrolysis and mild oxidation into a substantially pure vitamin.

2. Process for obtaining a purified antihemorrhagic compound comprising reacting a crude form of a compound of the class consisting of vitamins K1 and K2 with a reducing agent to obtain the corresponding hydroquinone-like compound, reacting the latter compound with an agent of the class consisting of esterifying and etherifying agents to obtain a compound of the class consisting of the corresponding esterified and etherified dehiydro derivatives of vitamins K1 and K2 and. hydrolyzing and oxidizing said last mentioned compound to obtain the original compound in a purified state.

3. Process for obtaining an antihemorrhagic compound comprising treating an impure form of a quinone compound having antihemorrhagic activity with a reducin agent and an agent of the class consisting of esterifying and etherifying agents to obtain a compound of the class consisting of the corresponding esterified and etherified hydroquinone compounds and hydrolyzing and oxidizing said last mentioned compound to obtain the original quinone compound in a more purified state.

4. Dihydro vitamin K2.

5. In a process for obtaining an antihemorrhagic compound, the step of mildly oxidizing a hydroquinone derivative of a 2- methyl-3-alkenyl-1, 4-naphthoquinone compound having antihemorrhagic atcivity, at its 1 and 4 positions only, to obtain said naphthoquinone compound.

6. In a process for obtaining an antihemorrhagic compound, the steps of hydrolizing and mildly oxidizing a compound of the class consisting of esterified and etherified hydroquinone derivatives of a 2-methyl-3-alkeny1-1,- 4-naphthoquinone compound having anti- 8 hemorrhagic activity,

EDWARD A. DOISY. SIDNEY A. THAYER. DONALD W. MACCORQUODALE. RALPH W. MCKEE. STEPHEN B. BINKLEY.

REFERENCES CITED The following references are of record in the file of this patent:

I UNITED STATES PATENTS Number Name- Date 1,984,511 Anderson Dec. 18, 1934 2,348,037 Thayer May 2, 1944 at its 1' and. 4' positions only, to obtain said naphthoquinone compound." 

